Protein therapeutics represent the most rapidly expanding class of drugs, allowing for the treatment of patients with diabetes, cancer, neurological diseases, anemia, infectious diseases, and immunological diseases, among others. Proteins in their natural state are folded into regions of secondary structure, such as helices, sheets, and turns. The α-helix is one of the most common structural motifs found in proteins, and many biologically important protein interactions are mediated by the interaction of an α-helical region of one protein with another protein. However, α-helices have a propensity for unraveling and forming random coils, which are, in most cases, biologically less active, or even inactive, and are highly susceptible to proteolytic degradation.
Several research groups have developed strategies for the design and synthesis of stabilized secondary structures. Some efforts have focused on helix-stabilizing side chain interactions or template-nucleated α-helix formation (Scholtz and Baldwin, Ann. Rev. Biophys. Biomol. Struct. 1992, 21, 95). Another approach has been to stabilize the helix via covalent crosslinks. However, the majority of the reported methodologies involve the use of polar and/or labile crosslinking groups, such as disulfide bonds (see, for example, Phelan et al. J. Am. Chem. Soc. 1997, 119, 455; Leuc et al. Proc. Nat'l. Acad. Sci. USA 2003, 100, 11273; Bracken et al., J. Am. Chem. Soc. 1994, 116, 6432; Yan et al. Bioorg. Med. Chem. 2004, 14, 1403). Verdine and colleagues have developed an alternative olefin metathesis-based approach, which employs α,α-disubstituted non-natural amino acids containing alkenyl side chains, which are subsequently “stapled” together using an olefin metathesis catalyst (Schafmeister et al., J. Am. Chem. Soc. 2000, 122, 5891; Blackwell et al., Angew. Chem. Intl. Ed. 1994, 37, 3281). These stapled peptides have been shown to resist proteolytic cleavage, and a stapled α-helical peptide derived from the BH3 helix of Bcl-2 has demonstrated utility in blocking the growth of leukemia cells in mice (Walensky et al., Science 2004, 305, 1466). In some cases, stapling can impart on the peptide the ability to enter cells through vesicular transport. Stapling can greatly increase in vivo half-life, most likely through binding to human serum albumin, and stapling can also increase the affinity for a receptor by as much as 103-104-fold.
Many proteins have α-helical segments that may benefit from covalent crosslinking to either stabilize the protein and/or alter a protein's biological activity. For example, the cytokine IL-13 has been identified as a therapeutic protein target, as it is strongly implicated in the pathogenesis of asthma. IL-13 is a soluble, secreted protein that folds to form a four-helix bundle structure (Moy et al., J. Mol. Biol. 2001, 310, 219; Eisenmesser et al., J. Mol. Biol. 2001, 310, 231). IL-13 signals by simultaneously engaging two transmembrane receptor subunits, IL-4Rα and IL-13Rα, thus causing receptor dimerization. IL-13 binding to the heterodimeric receptor triggers phosphorylation of the signal transducer and activator of transcription-6 (STAT-6), ultimately leading to an allergic response (Kelly-Welch et al., Science 2003, 300, 1527). IL-13 and its heterodimeric receptor are widely considered to be among the more attractive targets for treating asthma (Wills-Karp, Immunol. Rev. 2004, 202, 175).
Given the need for stabilized protein therapeutics, some of which are larger than can be produced synthetically, there remains a need in the art for the efficient synthesis of proteins with a stapled or stitched peptide segment. Such a technology would allow for the production of large quantities of proteins greater than 50 amino acids in length with a stapled or stitched peptide segment. There are many reasons for incorporating a stapled or stitched segment into a protein; some stapled or stitched proteins may be targeted to certain tissues or cells or taken up by cells through vesicular transport. Some proteins could be converted from an agonist to an antagonist through the incorporation of a staple. A protein could also gain new function via stapling.